PROJECT SUMMARY/ABSTRACT G-protein-coupled receptors (GPCRs) are a large and diverse class of cell surface receptors responsible for regulating nearly every physiological process in the human body. Upon binding to the extracellular surface of GPCRs, ligands trigger conformational changes that lead to binding and activating signaling proteins at the cytoplasmic surface of the receptor. Many GPCRs signal through several pathways that include different heterotrimeric G-protein subtypes as well as arrestins, and ?biased? ligands differentially modulate receptor signaling through these various pathways. GPCRs rely on a high degree of conformational flexibility to achieve this signaling complexity, and one of the major goals in the field of structure-based drug design is to identify the conformations that generate a particular signaling profile. Significant progress toward this goal has been made, yet relatively little is known about the effects of endogenous modulators, including protein-lipid interactions, post- translational modifications (PTMs), and accessory proteins, on GPCR structure and dynamics. The effect of endogenous modulators on the conformational landscape and the interplay of endogenous modulators with potential drugs need to be characterized to achieve the objectives of rational drug design, and this is a major long-term goal of my laboratory. In this project, we will focus on two PTMs of the ?2-adrenergic receptor (?2AR)? palmitoylation and glycosylation?with the goal of identifying the structural and dynamical basis for their regulation of ?2AR signaling. A complementary combination of continuous-wave and pulsed EPR techniques, mass spectrometry, and functional assays will be used in these studies. Importantly, cutting-edge variable- pressure EPR technology will provide unique mechanistic insights by mapping sparsely populated regions of the conformational landscape. First, we will map the conformational landscape at the cytoplasmic surface in the reference state of the receptor, defined herein as the natively glycosylated and palmitoylated receptor embedded in a lipid bilayer. Then, we will perform experiments with the PTMs removed, one at a time, to identify the allosteric effects of palmitoylation and glycosylation on the cytoplasmic surface. Finally, we will determine the local effects of glycosylation on the ligand-binding pocket at the extracellular surface and of palmitoylation on helix 8 and the C-terminal tail. The results will provide insight into the understudied yet critical role of these PTMs as regulators of ?2AR signaling, and because the ?2AR is a prototypical member of the GPCR superfamily, we anticipate the results to be applicable to other GPCRs with similar PTMs. In detailing the effects of endogenous modulators on the conformational landscape, our results will increase researchers' ability to rationally design drugs to achieve the desired therapeutic effect by allowing the in vivo interplay of endogenous modulators with drug candidates to be predicted more accurately.